EP2408914A1 - Method for determining the sensitivity or resistance of retrovirus isolates to molecules, therapeutic retroviral treatments based on viral protease inhibitors and diagnostic kits - Google Patents
Method for determining the sensitivity or resistance of retrovirus isolates to molecules, therapeutic retroviral treatments based on viral protease inhibitors and diagnostic kitsInfo
- Publication number
- EP2408914A1 EP2408914A1 EP10739694A EP10739694A EP2408914A1 EP 2408914 A1 EP2408914 A1 EP 2408914A1 EP 10739694 A EP10739694 A EP 10739694A EP 10739694 A EP10739694 A EP 10739694A EP 2408914 A1 EP2408914 A1 EP 2408914A1
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- European Patent Office
- Prior art keywords
- protease
- molecules
- retrovirus
- hiv
- seq
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
- C12N15/1034—Isolating an individual clone by screening libraries
- C12N15/1075—Isolating an individual clone by screening libraries by coupling phenotype to genotype, not provided for in other groups of this subclass
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
- C12N15/1034—Isolating an individual clone by screening libraries
- C12N15/1079—Screening libraries by altering the phenotype or phenotypic trait of the host
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/70—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving virus or bacteriophage
- C12Q1/701—Specific hybridization probes
- C12Q1/702—Specific hybridization probes for retroviruses
- C12Q1/703—Viruses associated with AIDS
Definitions
- This disclosure relates to methods for determining the sensitivity or resistance of retroviruses isolates to molecules, therapeutic retroviral treatments based on viral protease inhibitors, and diagnostic kits derived from the implementation of the methods.
- HIV-2 infectious virus
- HIV-I Aminodeficiency virus
- Lancet. 1 6-8; Marlink R, P Kandki, I Thior, K Travers, G Eisen, T Siby, I Traore, C C Hsieh, M C Dia and E H Gueye. 1994. Reduced rate of disease development after HIV-2 infection as compared to HIV-I.
- the plasma viral load of individuals infected by HIV-2 is less high and/or lower than that in individuals infected by HIV-I (Andersson S, H Norrgren, Z da Silva, A Biague, S Bamba, S Kwok, C Christopherson, G Biberfeld, and J Albert. 2000. Plasma viral load in HIV-I and HIV-2 singly and dually infected individuals in Guinea- Bissau, West Africa: significantly lower plasma virus set point in HIV-2 infection than in HIV-I infection. Arch. Intern. Med. 160:3286-93; Popper S J, A D Sarr, K U Travers, A Gueye-Ndiaye, S Mboup, M E Essex, and P J Kanki. 1999.
- HIV human immunodeficiency virus
- J Infect Dis. 180:1116-21. the individuals infected by HIV-2 develop the illness more slowly
- Vanittinghoff E S Scheer, P O'Malley, G Colfax, S D Holmberg and S P Buchbinder. 1999. Combination antiretro viral therapy and recent declines in AIDS incidence and mortality.
- HIV-2 was identified for the first time in West Africa in 1986 (Clavel F, D Guetard, F Brun-Vezinet, S Chamaret, M A Rey, M 0 Santos-Ferreira, A G Laurent, C Dauguet, C Katlama, and C Rouzioux. 1986. Isolation of a new human retrovirus from West African patients with AIDS. Science. 233:343-6).
- HIV-2 In this region, the prevalence of HIV-2 varies between 1% and 10% (Langley C L, E Benga-De, C W Critchlow, I Ndoye, M D Mbengue-Ly, J Kuypers, G Woto-Gaye, S Mboup, C Bergeron, K K Holmes, and N B Kiviat. 1996. HIV-I, HIV-2, human papillomavirus infection and cervical neoplasia in high-risk African women. AIDS. 10:413-7; Poulsen A G, B Kvinesdal, P Aaby, K Molbak, K Frederiksen, F Dias and E Lauritzen. 1989.
- the individuals infected by HIV-I and/or by HIV-2 are also treated by chemical therapy, composed of molecules having an inhibiting activity for the viral entry process or for the viral enzymes: Reverse Transcriptase, Protease, and Integrase.
- chemical therapy composed of molecules having an inhibiting activity for the viral entry process or for the viral enzymes: Reverse Transcriptase, Protease, and Integrase.
- that treatment has significantly helped to reduce morbidity and mortality caused by HIV infection (Palella F J, Jr, K M Delaney, A C Moorman, M O Loveless, J Fuhrer, G A Satten, D J Aschman and S D Holmberg. 1998. Declining morbidity and mortality among patients with advanced human immunodeficiency virus infection. HIV Outpatient Study Investigators. N. Engl. J. Med.
- genotyping approach Two different technical approaches were developed to orient therapeutic treatments. One consisted of searching only the already known mutations within the nucleic acid sequences coding for the viral proteins and is called the genotyping approach. The other one, that does not need knowledge of the presence of resistant mutations within the viral sequences, consists of testing in a cell based system the inhibition of viral replication in the presence of inhibiting molecules, and is called the phenotyping approach.
- a method of determining sensitivity or resistance of isolates of HIV retroviruses to a molecule including a) amplifying sequences coding for a protease of a retrovirus to be studied, with or without the or some of amino acid sequences situated upstream and downstream of a cleavage site of a precursor in which the amino acid sequences are situated, b) recombining fragments of DNA, a final product of the amplification, and an expression vector allowing expression of sequence coding for the protease of the retrovirus to be studied under control of a known inducible promoter through co-transformation of the vector and the DNA fragments with at least one yeast cell, c) culturing co-transformed yeast cell or cells to obtain a sufficient number of transformants to perform a sensitivity or resistance test, and recovering transformants issuing from the co-transformed cell, on any suitable medium, d) incubating the transformants in the presence of a molecule to be tested, e) qualitatively or
- nucleotide primers selected from the group consisting of SEQ ID NO 38, 39, 40, 41, 42, 48, 51, 52, 53, 54, 55, 56, 57 and 58; at least one expression vector; at least one strain of yeast; and at least one multi-well plate or other support.
- FIGS. 1 and 2 are schematic representations of the co-transformation of the pRS316-Gall/10M vector cleaved by the Notl restriction enzyme with the product of the second PCR to obtain a transformed yeast cell having the sequence of the HIV-2 protease under the control of the inducible promoter Gall.
- FIG. 3 is a schematic representation of the counting of living and dead cells and a determination of the phenotype.
- FIG. 4 is a schematic representation of a diagnostic kit.
- FIG. 5a shows a pair of graphs of percentage of living cells as a function of inhibitor concentration.
- FIG. 5b shows photos of cell growth as a function of resistance to inhibitor.
- DETAILED DESCRIPTION [0023] We provide methods for determining the sensitivity or resistance of retroviruses such as HIV to molecules, therapeutic treatments based on viral protease inhibitors, by the use of yeast. In other words, we provide for the use of yeast to determine the resistance or sensitivity of the viral protease to molecules, and/or the chemical molecules used in the context of therapeutic protocols. We also provide diagnostic kits comprising the elements necessary for implementing the method. [0024] More particularly, the methods make it possible to determine, quickly and at low cost, the resistance phenotype of the HIV-2 protease in infected patients.
- the methods also make it possible to determine, quickly and at low cost, the resistance phenotype of HIV-I protease in infected patients.
- the methods further allow the "high speed” and/or "high throughput” search for new chemical molecules having an inhibiting activity for the viral protease with a view to developing novel therapeutic treatments.
- sequences coding some therapeutic targets for example, reverse transcriptase protease, and integrase, as well as the so-called "structure proteins" (matrix, capsid, nucleocapsid), are situated within a common polypeptide precursor called Gag-Pol, coded by the gag-pol viral gene (Clavel F, Guyader M, Guetard D, Salle M, Montagnier L. Alizon M. 1986. Molecular cloning and polymorphism of the human immune deficiency virus type 2. Nature, 324:691-5).
- a side chain at position 48 of the human immunodeficiency virus type-1 protease flap provides an additional specificity determinant (Virology. 207:475-85, Boross P, Bagossi P, Copeland T D, Oroszlan S, Louis J M, Tozser J. 1999. Effect of substrate residues on the P2' preference of retroviral proteinases. Eur J Biochem. 264:921-9).
- a scientific article that appeared in 2003 demonstrated that the expression of the HIV-I protease by the yeast Saccharomyces cerevisiae caused the death of the latter through a still unknown mechanism, the consequence of which was the cell lysis of the yeast in question (Blanco R, Carrasco L and Ventoso I. 2003. Cell killing by HIV-I protease. J. Biol. Chem. 278: 1086-93).
- the smallest active protease precursor was defined as the smallest Gag-Pol sequence, containing the protease flanked by its cleavage sits that only induces yeast cell death when the cleavage site situated at the N-terminal part of the protease sequence is present. In the absence of this one, the expressed precursor is unable to disturb cell growth.
- our methods allow us to determine the sensitivity or resistance of retrovirus protease to molecules in a cell based non-infectious system in the presence of its natural substrate. Further, our methods allow us to test molecules having an effect on the activity of the protease and also on expression of the protease. Moreover, our methods allow us to test molecules having a direct effect on the activity of the protease toward its natural substrate that is, for example, the protease precursor.
- the method allows us to test molecules inhibiting the activity of protease by acting on the sequence coding for the protease, and/or coding for the protease precursor, by acting on the translation of the protease, by acting on the transcription mechanism, and/or on the protease activity.
- our methods allow for the determination of the sensitivity or resistance of protease having at least one mutation, for example, in the protease and the protease precursor coding nucleic acids sequence.
- the methods comprise an expression vector, choice of the cell system, method of expressing proteases of infected individuals and test of susceptibility to drugs.
- nucleic acids, DNA and/or RNA optionally extracting the nucleic acids, DNA and/or RNA from body fluids or cells (blood or other) taken from an individual or animal infected by the retrovirus, by any suitable means;
- the nucleic acids may be extracted from cells infected by a retrovirus, and/or body fluids from an infected individual or animal, and/or blood from an infected individual or animal, and/or from infected culture cell media.
- test molecules are selected from the group comprising molecules of a library, chemical molecules, natural molecules and molecules extracted from plants.
- the test molecules may be selected from the group comprising chemical molecules having an inhibiting activity on the viral protease, therapeutic treatments based on inhibitors of the viral protease or of the viral maturation.
- sequences of the protease amplified according to the method include those coding for the isolated protein or those coding for the protein and comprising all or some of the amino acid sequences situated upstream and downstream of the cleavage site of the protein precursor in which they are situated.
- the retrovirus may be HIV-I.
- the amplified sequences coding for the protease of the retrovirus to be studied according to the method can be mutated or non-mutated sequence.
- the amplified sequences coding for the protease of the retrovirus, for example, HIV-I, to be studied may be selected from the group comprising SEQ ID N° 10 to 37.
- the yeast used in the method may be of the type of Saccharomyces cerevisiae.
- the nucleic acids may be amplified, for example, by Polymerase Chain Reaction using couple of primers selected from the group comprising SEQ ID N° 38, 39, 40, 41, 42, 48, 51 to 58.
- the protocols used in the laboratory with a view to obtaining yeast transformants coding for an exogenous gene generally begin with a first step for obtaining the fragments of DNA coding for the gene of interest, either by gene amplification (Polymerase Chain Reaction (PCR) technique), or by release of the gene by virtue of the action of the restriction enzymes, which cuts the DNA containing the sequence of interest.
- This first step is equivalent in time to half a day's work.
- the DNA fragment released is then sub-cloned in an expression vector by the action of the DNA Ligase enzyme (an operation lasting one night) and the product of the reaction is amplified in a bacterium, after its transformation (one day to obtain bacteria having incorporated plasmid DNA, and one and a half days of obtaining and characterizing the transform ant sought, containing the plasmid coding for the gene of interest).
- the bacterial clone obtained in the previous step may be amplified (one night) and the plasmids purified by conventional known methods (one day).
- the purified plasmid obtained is then used to transform the selected yeast strain (1/2 day).
- the transformed yeast strain is obtained approximately 4 days after the transformation event.
- the culture of the transformants can be done, for example, in liquid medium and/or solid medium.
- the medium used in our methods can be any known medium and suitable for the culture of the transformants, for example, minimal synthetic media containing a carbon source such as glucose or galactose and lacking or not a specific nutrient, for example, uracil.
- Qualitative or quantitative analysis means any analysis of living cells known in the art. For example, it can be a step of counting or scoring the living cells, for example, by measuring the absorbance, for example, at 600 nm of a liquid medium, or by eye counting, by observation when the transformants are cultured, for example, on a solid medium. It can also be, for example, the result of laboratory testing for the sensitivity of the transformants to a test molecule or another method involving the use of a test molecule.
- molecule to be tested diffuses in the area surrounding each disc. It can also be, for example, a quantitative way based on dilution: a dilution series of molecule to be tested is established, that is, for example, a series of reaction vials with progressively lower concentrations of molecule substance.
- the sensitivity or resistance of protease can be deduced, for example, by measuring the half maximal inhibitory concentration (IC50), by comparing the living number of cells between transformants incubated with molecules and non-incubated with molecules.
- IC50 half maximal inhibitory concentration
- This technique very advantageously simplifies obtaining the transformants by reducing in particular the number of manipulations which involve a significant reduction in the experimentation time necessary (approximately half compared to known protocols) and in production cost. Use of this technique also enables a large number of samples to be manipulated at the same time.
- protease expressed is the isolated protein.
- the primers and nucleotide fragments disclosed above will be modified accordingly.
- the aim of the modification is to be able sub-clone the gene of the protease from virus infecting each infected individual studied and transform the yeast with the protease gene obtained, by a simple and rapid procedure (a single-step procedure).
- pRS316-Gall/10 which comprises the inducible promoter GAL 1/10 in position 5' of the cloning site of the gene to be expressed (Liu H, Krizek J, and Bretscher A. 1992. Construction of a GAL-I regulated yeast cDNA expression library and its application to the identification of genes whose overexpression causes lethality in yeast. Genetics 132:665-673).
- the viral gene is expressed when the cell is transformed with this vector in the presence of Galactose and the gene is not expressed when the cells transformed are in the presence of glucose as a carbon source.
- the vector For the fragment of amplified viral DNA to be able to be inserted by homologous recombination in the expression vector, the vector must be modified by adding to it, just after the sequence of the inducible promoter, a primer 5' of approximately 40 pairs of bases, followed by a single restriction site (to be able to linearize the vector), and a primer 3' of approximately 40 pairs of bases.
- this modified and linearized vector is a good substrate for the homologous recombination event, the sequence introduced at position 5' (between the gene and the promoter) must not inhibit the transcription of the genes.
- pRS316-Gall/10 expression vector thus modified is called pRS316-Gal 1/1 OM. 2 ⁇ Choice of the Cell System
- the DNA sequence coding the HIV-2 protease is amplified by the PCR technique, twice. [0084] The first amplification produces a significant quantity of DNA. This reaction takes place using DNA extracted from lymphocytes of the peripheral blood of infected individuals.
- the nucleotide primers used are of the type:
- Sense primer 5'-GAAAGAAGCCCCGCAACTTC-S' (SEQ ID N° 3)
- Antisense primer 5'-GGGATCCATGTCACTTGCCA-S'
- the second amplification frames the protease of an initialization codon of the transcription (ATG) and of a termination codon of the transcription (TAA) and adds on each side the sequences approximately 40 nucleotides that we brought to the vector when it was modified.
- This PCR reaction takes place on the product of the first PCR with the primers of the type:
- Antisense primer 5'GCGGAGCTCGCTTTAGCATTATTTTTATTGGCTCTACTGCGGCCGCTTAAG
- Framing the protease by the initiation and termination codons is that of the isolated protease or the protein comprising all or some of the amino acid sequences situated upstream and downstream of the cleavage site of the precursor in which it is situated.
- a sample of peripheral blood is taken from an individual infected with HIV-2.
- the lymphocytes issuing from this sampling are purified or not, and their DNA extracted by known methods.
- the DNA undergoes the two aforementioned PCR reactions to create the fragment of DNA, carrying the sequence of the protease with or without the or some of the amino-acid sequences situated upstream and downstream of the cleavage site of the precursor in which it is situated and compatible with the expression vector to cause the phenomenon of homogeneous recombination in the transformed cells.
- a yeast strain having a genotype ura3 is co-transformed with the pRS316Gall/10M linear vector (by its Notl site).
- the transformants potentially producing the protease are recovered on any suitable carrier such as, for example, gelose composed of agar, glucose as a source of carbon, and a synthetic environment with a deficiency of uracil.
- the test was also done on plasma issuing from this sampling, purified or not, and their RNA or DNA extracted by known methods. In this case, these nucleic acids undergo the aforementioned RT-PCR and PCR reactions, if they are of RNA type, and no RT phase if they are of type DNA. [0096] The interval of time between the blood sampling and the definition of the resistance profile is only one week.
- Example 2 [0097] In this example, the expression vector was modified as follows:
- a first modified version of the vector pRS316-Gall/10 was created by exchanging the BamHl-Sacl fragment of this vector with a DNA fragment, also framed by the BamHl-Sacl restriction sites that contains (from 5' to 3'):
- a second modified version of the vector pRS316-Gall/10 was created by exchanging the BamHl-Sacl fragment of this vector with a DNA fragment, also framed by the BamHl-Sacl restriction sites that contains (from 5' to 3'):
- Any strain of yeast with necessary auxotrophy markers to allow the selection of the transformant expressing the viral protease and preferably any strain of Saccharomyces cerevisiae.
- Saccharomyces cerevisiae was used.
- nucleic acid sequence coding the HIV protease, or any of its precursor forms were amplified by the RT-PCR technique, followed by a PCR reaction as in Example 1.
- nucleotide primers used for the RT-PCR reaction were of the type:
- Primer R2139S1 GAGCTCTTAATCCATTCCTGGCTTTAATTTTAC TGGTACAGTTTCAATTGGAC, and
- Primer5T-pc-rh TATACTTTAACGTCAAGGAGAAAAAAAACCCCGGA
- Primer5'F3 TATACTTTAACGTCAAGGAGAAAAAACCCCGGAT
- the co-transformation of the pRS316-Gall/10M-3 vector cleaved by the BamHl restriction enzyme with the product of the second PCR, performed with primers SEQ ID N° 42 and 55 makes it possible, through the homologous recombination event that took place in the cell, to obtain a transformed yeast cell, carrying the sequence of a precursor form of the HIV-I protease that consists of the protease coding sequence followed by 13 amino acids, under the control of the inducible promoter Gal.
- a sample of peripheral blood is taken from an individual infected with HIV-I.
- the plasma or lymphocytes issuing from this sampling are purified or not, and their RNA or DNA extracted by known extraction kits, like QlAamp Viral RNA Mini Kit from QIAGEN (The Netherlands).
- a yeast strain having a genotype ura3 is either co-transformed with the pRS316Gall/10M-3 linear vector (by its BamHl site) or co-transformed with the pRS316Gall/10M-4 linear vector (by its Xhol site).
- the transformants potentially producing the protease are recovered on any suitable carrier such as gelose composed of agar, glucose as a source of carbon, and a synthetic environment with a deficiency of uracil.
- the integral protease precursor sequence was amplified by PCR from the laboratory viral pNL4.3 DNA strain with using the following primers:
- the PCR product (SEQ ID N° 18) was purified by standard procedures digested by restriction enzyme Xbal (Invitrogen, USA), following the manufacturing recommendations and introduced, through overnight DNA ligation (T4 DNA Ligase from Gibco USA), as manufacturing recommendations into previously Xbal digested pRS316-Gal/10 vector.
- Xbal restriction enzyme
- 2 Protease Precursor Sequence Starting at the Beginning of the Protease Coding Sequence and Ending at the End of the Pol Coding Region (SEQ N°
- PCR reaction was performed on purified DNA construct in pRS316-Gal/10 vector obtained in Item 1. PCR conditions and other experiences were the same as conditions described in previous Item 1.
- protease precursor sequence from protease coding sequence until the end of the gag-pol coding sequence was obtained using the following primers:
- the primers were selected to provide sequences situated on both sides of the cloning site in the expression vector.
- Plasmid containing the gag-pol precursor sequences starting at the protease and ending at the end of the pol region was linearized by digestion with restriction enzyme Xhol (Invitrogen, USA) according to manufacturing recommendations.
- Antisens M13-gp-rh ATTATCCATTATCCATTCTCGAGGTTAA
- oligonucleotides further contain initiation and termination codons and identical sequences to the pRS316-Gall/10 vector.
- primer 5'-a435 CAGGCTAATTTTTTAAGGG
- primer R-a435 CCCTTAAAAAATTAGCCTG
- primer F 1071 B 1 GGATCC ATGATGAC AGC ATGTC AGGGAGTGGGAG
- the mutated precursor was made in two steps of PCR.
- 2 independent reactions using as template the construct obtained in previous Item 2, involved pairs of primers, SEQ ID N° 49 and SEQ ID N° 55 for one and SEQ ID N° 50 and SEQ ID N° 51 for the second, to introduce a further A (Adenine) at position 1638 of the sequence.
- HIV-I protease precursor form coding sequence was amplified from purified total RNA extracted from blood plasma of infected patients by RT-PCR followed by a PCR reaction, as described in Example 2.
- the primers used for the RT-PCR reaction were: (SEQ ID N° 52)
- the second amplification frames the protease between an initialization codon of the transcription (ATG) and a termination codon of the transcription (TAA).
- ATG was situated 70 nucleotides above of the first codon of the protease.
- TAA was situated 150 nucleotides below of the last codon.
- the second amplification adds on both sides sequences corresponding to the vector to clone it in yeast by homologous recombination. This PCR was made on the first PCR product with the following primers:
- Primer R2139S1 GAGCTCTTAATCCATTCCTGGCTTTAATTTTA
- Example of Determining the Resistance/Sensitive Character of a Protease from HIV-I Infected Individual An HIV-I short protease precursor form, of the size of SEQ ID N° 15, coding sequence was amplified by RT-PCR followed by a PCR reaction, as described in Example 2, from purified total RNA extracted from blood plasma of an infected patient, namely P#10, known to carry HIV-I strains resistant to protease inhibitors like lopinavir, saquinavir and darunavir.
- Primer 3' R772 CCTGAAAATCCATAYAAYAC, for the RT-PCR step, and the primers: (SEQ ID N° 55)
- Primer R2139S1 GAGCTCTT AATCCATTCCTGGCTTTAATTTT
- Primer 5' F-pc-rh ATACTTTAACGTCAAGGAGAAAAAACCCCGGA TCCATGTGGGGTAGAGACAACAACTCC, for the second amplification.
- the obtained final PCR product was cloned into pRS316-Gall/10M-4 by the co- transforming single step procedure described in Example 2 (3).
- the transformed yeast cell was of the strain BY4741 (Euroscarf, Germany) having the following genotype: MATa, his3. ⁇ .l, leu2. ⁇ .O, metl5. ⁇ .O, ura3. ⁇ .O.
- the procedure described in Example 3 (5) was performed to obtain yeast cells expressing sensitive and resistant proteases.
- the sensitive or resistant character of the cloned protease was determined in liquid and in solid media.
- the higher concentration of the molecules tested were:
- the viral protease from patient P#10 is resistant to the tested inhibitor molecules.
- the yeast transformant expressing each viral protease was plated on synthetic minimal media, agar plate, in the presence of 2% Galactose.
- a 5 mm diameter paper disk (Minitrans-Blot, Bio-Rad USA) impregnated with 10 microliters of either 0.5 milHMolar lopinavir, 0.5 m milliMolar darunavir, or 1 milliMolar saquinavir, was placed in the center of the plate. After 3 days at 30.degree. C. observation by naked eye was performed.
- the expressed viral protease was sensitive to the inhibitor, a halo of growing cells was observed, whereas no growing cells were observed when the viral protease expressed in transformed yeasts were resistant to inhibitor (FIG. 5b).
- the method allows determination of the sensitivity or resistance of isolate of HIV retrovirus to molecules.
- a diagnostic kit also determines the sensitivity or resistance of retrovirus isolates to therapeutic retroviral treatments based on inhibitors of the viral protease. It may comprise:
- nucleotide primers and, for example, nucleotide primers as previously disclosed for implementation of the amplification of DNA coding for the retrovirus protease by the PCR technique namely:
- Sense primer 5'-GAAAGAAGCCCCGCAACTTC-3 1 (SEQ ID N° 3)
- Antisense primer 5'GGGATCCATGTCACTTGCCA-S' [0166] for the second amplification, primers of the type: (SEQ ID N° 4)
- Antisense primer 5 'GCGGAGCTCGCTTTAGCATT ATTTTTA
- At least one expression vector and, for example, the plasmid modified and linearized according to our methods and as previously described, for example, the plasmid pRS316-Gall/10M, M-3, M-4;
- At least one multi-well plate or any other suitable support At least one multi-well plate or any other suitable support.
- the primers and nucleotide fragments will be modified accordingly.
- a principle of the kit is as follows: [0172] Using the blood of the patient, the gene coding for the viral protease is amplified by an RT-PCR reaction with the primers described in the method and supplied in the kit.
- Amplification of the gene of the viral protease using cell DNA is also possible.
- the amplified product and the modified and linearized vector are used for transforming a strain of yeast, also supplied in the kit, in a multi-well plate.
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US50317409A | 2009-07-15 | 2009-07-15 | |
US12/504,456 US9493769B2 (en) | 2004-06-02 | 2009-07-16 | Yeast-based assay for measuring the functional activity of an HIV-1 protease in response to an antiviral agent |
PCT/IB2010/001738 WO2011007244A1 (en) | 2004-06-02 | 2010-07-14 | Method for determining the sensitivity or resistance of retrovirus isolates to molecules, therapeutic retroviral treatments based on viral protease inhibitors and diagnostic kits |
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2010
- 2010-07-14 CA CA2767955A patent/CA2767955A1/en not_active Abandoned
- 2010-07-14 JP JP2012520118A patent/JP5785165B2/en not_active Expired - Fee Related
- 2010-07-14 EP EP10739694.7A patent/EP2408914B1/en not_active Not-in-force
- 2010-07-14 ES ES10739694T patent/ES2426024T3/en active Active
Non-Patent Citations (1)
Title |
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See references of WO2011007244A1 * |
Also Published As
Publication number | Publication date |
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JP5785165B2 (en) | 2015-09-24 |
ES2426024T3 (en) | 2013-10-18 |
JP2012532625A (en) | 2012-12-20 |
EP2408914B1 (en) | 2013-06-12 |
CA2767955A1 (en) | 2011-01-20 |
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